Chlamydomonas mutants produced using RGEN RNP and method for preparing pigment using the same

ABSTRACT

A new alga having an improved ability to produce a pigment is disclosed. When the alga is used, a carotenoid-based pigment, specifically, a xanthophyll can be produced by consuming less energy, so that it is possible to efficiently produce the pigment at the industrial level. The pigment can be applied as a raw material for a food, a health functional food and a medicine, which include the pigment. Since a DNA fragment is not likely to be inserted into a target base sequence or a base sequence other than the target, it is expected that the procedure of constructing the mutant is not regulated as a GMO.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No. 16/050,012 filed on Jul. 31, 2018, which is a Continuation-in-Part (CIP) of PCT Application No. PCT/KR2017/004268, filed on Apr. 21, 2017, which claims priority to Korean Patent Application No. 2016-0049439, filed Apr. 22, 2016, and to Korean Patent Application No. 2017-0041761, filed Mar. 31, 2017, and to Korean Patent Application No. 2017-0041762, filed Mar. 31, 2017, the disclosures of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an alga having a pigment producing ability, a dye composition containing the alga, and a method for preparing the pigment.

BACKGROUND ART

Macular degeneration is a disease in which degeneration occurs in the macula which is a nerve tissue located in the center of the inner retina of the eye and causes vision impairment, and since most of the photoreceptors are gathered in the macula and a place where an image of an object is formed is also at the center of the macula, the macula plays a very important role in vision. The most common cause of macular degeneration may be an increase in age (age-related macular degeneration), and is known to be related to family history, race, and smoking. Because the macula is responsible for central vision, if degeneration occurs in the macula, a decrease in vision, central scotoma, metamorphopsia which is a symptom in which things appear distorted, and the like occur. Macular degeneration is largely classified into non-exudative (dry) and exudative (wet), and the non-exudative macular degeneration does not significantly affect vision in most cases, except for the late stage when the atrophy of the retina and the choroid appears, and is a step in which yellow deposits called drusen are seen under the retina, but in the case of the exudative macular degeneration in which subretinal hemorrhage or subretinal fluid, pigment epithelial detachment, and the like appear, when the position of such a lesion is present under the macula or immediately adjacent to the macula, a drop in vision appears from the initial stage. The exudative macular degeneration accounts for about 10 to 20% of the total cases of macular degeneration, but if the exudative macular degeneration is left as it is without being treated, vision rapidly deteriorates, so that many patients will be blind within two years after being diagnosed with the exudative macular degeneration. In order to prevent macular degeneration, it is important to find an abnormality of the macula early through a periodic funduscopic examination and to make an effort so as to reduce the adjustable factors such as obesity, smoking, and hypertension. Since smoking causes damage to choroidal circulation leading to a drop in antioxidant factors in blood and causes choroidal vasoconstriction to cause low oxidative damage, a patient who is at risk of macular degeneration necessarily needs to quit smoking. Further, since a macular pigment (lutein, zeaxanthin) reduces damage caused by aging and serves to maintain a healthy retina, sufficiently ingesting the macular pigment through vegetables and fruits or taking commercialized vitamin supplements can help in the prevention of macular degeneration.

The macular pigment serves to reduce age-related failing eyesight caused in the central part of the retina and prevent retinal tissue damage due to bright light, and representative examples thereof include a xanthophyll as a carotenoid-based oxycarotenoid pigment produced by oxygenation of a carotenoid. Examples of a pigment belonging to xanthophylls include lutein, zeaxanthin, or the like. It is known that lutein acts as an antioxidant that protects the inside of the eyes that is damaged by free oxygen radicals naturally produced in the body, reduces the growth of blood vessels that supply carcinomas to kill cancer cells, and has some effects on prevention of breast cancer, colon cancer, lung cancer, ovarian cancer, and skin cancer.

Animals cannot produce xanthophylls and can obtain xanthophylls only through ingestion of food, but these xanthophylls are present together with chlorophylls and carotenes in the green parts such as leaves, flowers, and fruits of plants. Recently, a health functional food for eye health, including xanthophylls, and the like has attracted attention.

Existing marigold flowers are representative as a raw material for zeaxanthin and lutein, and those extracted from other higher plants has also been studied. In addition, zeaxanthin and lutein are also produced by genetically mutating the pigment synthesis mechanism in bacteria. Studies have also been conducted to obtain these pigments from microalgae. Among these conventional raw materials, marigold flowers have a disadvantage in that it takes a long time to breed flowering plants for production, and have a problem in that the production unit cost is high because the production amount is not large as compared to the land area for production.

In order to solve these problems, the development of zeaxanthin and lutein-producing algae into which a pigment synthesis mechanism is inserted using a bacterial system for replacing a higher plant system was carried out, but there is a problem in that a pigment obtained from bacteria is not suitable for ultimate use as a food additive. In addition, since genetically modified organisms (GMOs) using a genetic insertion technology and the like are not preferred in the domestic market, the GMOs act as a fatal disadvantage in the food additive marker where consumers' perceptions are important, and likewise as in the higher plant system, there is a problem in that a large cost of maintaining a bacterial culture solution, a bioreactor, or the like may be required.

In the case of a method of obtaining these pigments from microalgae, the conventional microalgae are a wild type which is not improved, and have a limitation in being used as optimal producing algae because the content of lutein is constant, but the content of zeaxanthin is very low depending on the amount of light.

REFERENCES OF THE RELATED ART Patent Document

Korean Patent Application No. 2014-7007656

DISCLOSURE Technical Problem

An object of the present invention is to provide a method capable of replacing a xanthophyll used as a raw material of a conventional food or a method capable of replacing a conventional raw material production method, and specifically to provide a microorganism having an excellent ability to produce xanthophylls, particularly, lutein and zaexanthin, a composition including the same, and a method for preparing xanthophylls using the same.

Technical Solution

In order to achieve the aforementioned object, the present inventors have made efforts to develop algae capable of solving the insufficient productivity of wild-type or conventionally present microalgae by using other mutations without a genetic recombination method which may be a problem in the food industry, and as a result, developed a mutant having a higher yield of macular pigment than a conventional Chlamydomonas reinhardtii alga and identified an optimal method for preparing a pigment using the same, thereby completing the present invention.

In this regard, the present invention provides a Chlamydomonas reinhardtii mutant having a ZEP gene mutation in which a base sequence represented by SEQ ID NO: 2 is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1 and having an ability to produce xanthophylls.

Further, the present invention provides a Chlamydomonas reinhardtii mutant having a ZEP gene mutation in which a base sequence represented by SEQ ID NO: 4 is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1 and having an ability to produce xanthophylls.

In addition, the present invention provides a Chlamydomonas reinhardtii mutant having a ZEP gene mutation in which a base A is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1 and having an ability to produce xanthophylls.

The three Chlamydomonas reinhardtii mutants may each have an ability to produce xanthophylls.

The three Chlamydomonas reinhardtii mutants may each have an ability to produce one or more pigments selected from the group consisting of lutein and zeaxanthin; and chlorophyll b, chlorophyll a, and β-carotene.

Furthermore, the present invention provides a culture of the Chlamydomonas reinhardtii mutant.

Further, the present invention provides a pigment composition including one or more selected from the group consisting of a culture of the mutant, a dry material thereof, and an extract thereof.

In addition, the present invention provides a composition for oral administration, including one or more selected from the group consisting of a culture of the mutant, a dry material thereof, and an extract thereof.

Furthermore, the present invention provides a composition for feed or a feed additive, including one or more selected from the group consisting of a culture of the mutant, a dry material thereof, and an extract thereof.

Further, the present invention provides a composition for a food or food additive, including one or more selected from the group consisting of a culture of the mutant, a dry material thereof, and an extract thereof.

In addition, the present invention provides a method for preparing a pigment using the mutant.

Furthermore, the present invention provides a method for preparing a food or feed raw material, including: culturing the mutant.

Advantageous Effects

Through the present invention, it could be confirmed that three mutants were constructed by using the CRISPR gene scissors technology (RGEN RNPs) without any introduction of an exogenous DNA in a microalga Chlamydomonas reinhardtii to knock out a ZEP gene, and the amount of zeaxanthin which is an industrially useful pigment was significantly increased when cellular characteristics of the existing wild type and the three mutants of the present invention were compared with each other. In particular, since a DNA fragment is not likely to be inserted into a target base sequence or a base sequence other than the target, it is expected that the procedure of constructing the mutant is not regulated as a GMO, so that it is expected that the procedure of constructing the mutant can create a big economic effect in terms of an industry which produces lutein and zeaxanthin by using microalgae.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

FIG. 1 illustrates a general summary on the techniques and methods applied in the present invention;

FIG. 2 illustrates information on a zeaxanthin epoxidase (ZEP) gene of Chlamydomonas reinhardtii cw15 wild type (SEQ ID NO: 1);

FIG. 3 is a summary of five target sequences designed for targeting the Chlamydomonas reinhardtii ZEP gene (CACCAGCTGCGCGACCGAGCTGG, SEQ ID NO: 12; GCCGTTGCACTTCTGAAGCAGGG, SEQ ID NO: 13; TCCGGCGAACGCACCTGGATGGG, SEQ ID NO: 14; TGGTGGGCGCCGACGGCATCTGG, SEQ ID NO: 15; CCATGGCTTCGCAGGCATCTCGG, SEQ ID NO: 16) [5 sgRNAs were carefully designed within the half of a coding sequence region of a ZEP gene which is different from any other target sites by 3 nucleotides (nt) in the entire genome and has an out-of-frame score higher than 66 by using Cas-Designer (www.rgenome.net/cas-designer/). The ‘coding sequence (CDS) position’ refers to a relative position of an excision point in a RNA transcript. a + direction refers to a direction which is the same as a target sequence, that is, means that the same sequence is a sequence of RGEN, and − refers to a direction reverse to the target sequence, that is, a sequence having a reverse complement relationship with each other, which is a sequence bound to the target sequence. The out-of-frame score’ indicates the probability of a frame shift-inducing deletion occurring when a cleaved double-stranded DNA is repaired by a microhomology-mediated end joining (MMEJ) pathway. The ‘# of a target-off site’ refers to the number of sequences mismatched throughout the entire genome. The linking of the remaining sgRNA sequence (gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc) (SEQ ID NO: 10) to the target sequence results in the entire sgRNA];

FIG. 4 illustrates mutations of a ZEP gene induced by DNA-free RGEN RNPs [a: RGEN-transfected cell and wild-type mutation (insertion and deletion; indel) frequencies for each sgRNA were measured by targeted deep sequencing. The Indel frequency was measured up to about 0.46%. b: a representative mutant DNA sequence (RGEN3) obtained from the third sgRNA having the highest efficiency observed from the targeted deep sequencing analysis result of a, that is, TCCGGCGAACGCACCTGGATGGG (SEQ ID NO: 11). Various indel patterns identified from the target sequence by the targeted deep sequencing analysis result appearing at the 3 nt upstream of the PAM sequence. The 20-bp target sequence was underlined and the PAM sequence was indicated in bold.]. The depicted nucleotide sequences, listed from top to bottom, are SEQ ID NOs: 17-22;

FIG. 5 illustrates the Cas9 protein sequence used in Example 1 (SEQ ID NO: 9);

FIG. 6 is a set of photographs illustrating morphological characteristics of a Chlamydomonas reinhardtii cw15 wild-type alga and Chlamydomonas reinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 [a: measurement of chlorophyll (Chl) fluorescence for several hundreds of colonies to study ZEP gene knock-out, b: a photograph of cultivation in a colony state in a solid TAP medium containing agar, c: a photograph illustrating a state when the concentration was adjusted to the same concentration (OD 750=1) after a Chlamydomonas reinhardtii cw15 wild-type alga and Chlamydomonas reinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 were liquid-cultured in an HS medium];

FIG. 7 identifies variations in target DNA sequences of actual ZEP gene positions in the three ZEP mutants generated by DNA-free RGEN RNPs [a: wildtype (SEQ ID NO: 23), b: ZEP mutant 1 (ΔZ1) (SEQ ID NO: 24), c: ZEP mutant 2 (ΔZ2) (SEQ ID NO: 25), d. ZEP mutant 3 (ΔZ3) (SEQ ID NO: 26)];

FIG. 8A illustrates information on the ZEP gene of the Chlamydomonas reinhardtii mutant ΔZ1 (SEQ ID NO: 3);

FIG. 8B illustrates information on the ZEP gene of the Chlamydomonas reinhardtii mutant ΔZ2 (SEQ ID NO: 5);

FIG. 8C illustrates information on the ZEP gene of the Chlamydomonas reinhardtii mutant ΔZ3 (SEQ ID NO: 6);

FIG. 9 illustrates autotrophic culture vessels;

FIG. 10 illustrates mixotrophic culture vessels;

FIG. 11 is a set of HPLC analysis graphs illustrating pigment profiles of the Chlamydomonas reinhardtii cw15 wild-type and the Chlamydomonas reinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 [neo+lor: neoxanthin+loroxanthin, vio: violaxanthin, an: antheraxanthin, lut: lutein, zea: zeaxanthin, chl a: chlorophyll a, chl b: chlorophyll b, α-car: α-carotene), and β-car: β-carotene];

FIG. 12 is a graph illustrating the growth curves (the number of cells per volume, cells/ml) of the Chlamydomonas reinhardtii cw15 wild-type and the Chlamydomonas reinhardtii mutants ΔZ1, ΔZ2, and ΔZ3 over time;

FIG. 13 is a set of graphs comparing the amounts of lutein and zeaxanthin pigments produced by the Chlamydomonas reinhardtii cw15 wild-type (WT) and the ZEP knock-out mutants ΔZ1, ΔZ2, and ΔZ3 over time [a: the amount (mg/L) of lutein produced over time, b: the amount (mg/L) of zeaxanthin produced over time, c: the sum (mg/L) of the amounts of lutein and zeaxanthin produced over time]; and

FIG. 14 compares the contents of zeaxanthin and lutein among higher plants known to have high contents of zeaxanthin and lutein, the Chlamydomonas reinhardtii cw15 wild-type (WT), and the Chlamydomonas reinhardtii ZEP knock-out mutants ΔZ1, ΔZ2, and ΔZ3.

BEST MODE

Exemplary embodiments of the present invention will be described in detail below with reference to the accompanying drawings. While the present invention is shown and described in connection with exemplary embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made without departing from the spirit and scope of the invention.

However, the present invention may be modified in various forms and may have various forms, so that specific examples and descriptions set forth below are included merely for aiding the understanding of the present invention, and are not intended to limit the present invention to a specific disclosure form. It should be understood that the scope of the present invention includes all the modifications, equivalents, and replacements falling within the spirit and technical scope of the present invention.

Hereinafter, the present disclosure will be described in more detail.

The present relates to a Chlamydomonas reinhardtii mutant.

The Chlamydomonas reinhardtii is a eukaryote distributed in various environments such as fresh water and oceans as a unicellular green alga (Chlorophyta), and has a doubling time of 6 to 8 hours. Further, the Chlamydomonas reinhardtii is one of the microalgae model systems most widely distributed and can be produced in a bioreactor.

The mutant was constructed by using RGEN RNPs which is not a general mutation treatment, that is, the CRISPR gene scissors technology in which exogenous DNAs are not introduced to knock out zeaxanthin epoxidase (ZEP) genes.

The mutant is a mutant (hereinafter, referred to as ΔZ1) having a ZEP gene mutation in which a base sequence (gaaattaata agactcatta tattccggcg aacgcacctg ga) represented by SEQ ID NO: 2 is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1. That is, the mutant is a mutant having a ZEP gene mutation represented by SEQ ID NO: 3.

Further, the mutant is a mutant (hereinafter, referred to as ΔZ2 having a ZEP gene mutation in which a base sequence (tagctctaaa acatccaggt gcgttcgccg gactatagtg agta) represented by SEQ ID NO: 4 is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1. That is, the mutant is a mutant having a ZEP gene mutation represented by SEQ ID NO: 5.

In addition, the mutant is a mutant (hereinafter, referred to as ΔZ3) having a ZEP gene mutation in which a base A is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii cw15 WT represented by SEQ ID NO: 1. That is, the mutant is a mutant having a ZEP gene mutation represented by SEQ ID NO: 6.

The three Chlamydomonas reinhardtii mutants of the present invention each have an ability to produce a pigment, specifically, an ability to produce xanthophylls. Specifically, the three Chlamydomonas reinhardtii mutants of the present invention may have an ability to produce lutein and zeaxanthin. More specifically, the three Chlamydomonas reinhardtii mutants of the present invention may have an ability to produce one or more pigments selected from the group consisting of lutein and zeaxanthin; and chlorophyll b, chlorophyll a, and β-carotene.

Since the mutant has a significantly high ability to produce zeaxanthin per cell as compared to the conventional Chlamydomonas reinhardtii cw15 wild-type and a content of lutein and zeaxanthin which is more than 12 times higher than those of higher plants known to have a high content of lutein and zeaxanthin [see FIG. 14], there is an advantage in that the mutant can be effectively used as an alga for producing xanthophyll.

In a specific exemplary embodiment, it was confirmed that the mutant of the present invention had a significantly increased amount of zeaxanthin produced over time as compared to the Chlamydomonas reinhardtii cw15 wild-type (FIG. 13B), so that it could be seen that the mutant of the present invention had mycological properties having an excellent ability to produce xanthophylls, particularly, zeaxanthin, and it was confirmed that the mutant of the present invention could be effectively utilized as a source of producing a xanthophyll pigment by utilizing the mycological properties.

The mutant of the present invention can survive in dim light, and may be cultured under light intensity conditions specifically within a range of 10 to 2,000 μmol photons/m²s. The mutant cannot photosynthesize in complete darkness which is equal to or less than the dim light conditions, and cell can be damaged by lighting stress under excessive lighting conditions. When the mutant of the present invention is cultured under the conditions, there is an advantage in that the mutant of the present invention has an excellent growth rate while increasing the content of a xanthophyll in the mutant.

The mutant can be appropriately grown within typical growth environments (light intensity conditions, temperature conditions, medium, and the like) of a Chlamydomonas reinhardtii alga. Furthermore, since the mutant has an excellent ability to accumulate zeaxanthin even under low light intensity (FIG. 13), the mutant can be industrially and effectively as a xanthophyll pigment-producing microorganism due to the excellent ability to produce a xanthophyll, and the density thereof in a cluster is relatively lower than other algae even under high light intensity, so that the mutant has an effect of having an excellent efficiency of producing a pigment by photosynthesis in a single cell. Specifically, the Chlamydomonas reinhardtii wild-type produces almost no zeaxanthin, but the mutant of the present invention has a content of zeaxanthin, which is higher by about 50 times or more than that of the wild-type.

The mutant can be cultured in an environment capable of culturing a general Chlamydomonas reinhardtii alga, and specifically, it is possible to use a culture medium capable of culturing an alga under weak light intensity conditions. In order to culture a specific microorganism, the culture medium contains nutritional materials required by a subject to be cultured, that is, a microorganism to be cultured, and may be a culture medium in which a material for a special purpose is additionally added and mixed. The medium also refers to a culture medium or a culture solution, and is a concept encompassing all of the natural medium, the synthetic medium or the selective medium. The Chlamydomonas reinhardtii mutant may be cultured according to a typical culture method. For example, the Chlamydomonas reinhardtii mutant may be cultured in an HS medium or a TAP medium, which is a photosynthesis medium, and a carbon source may be added. In an exemplary embodiment, it was confirmed that in the culture solution composition environments in Table 1 in the Examples of the present invention, the mutant of the present invention had an excellent ability to produce zeaxanthin.

A pH of the culture medium is not particularly limited as long as the pH is within a range enabling Chlamydomonas reinhardtii to survive and be grown, and as an example, a pH of 6 or more, specifically, Chlamydomonas reinhardtii can survive within a PH of 6 to a pH of 9, and may have an optimal growth rate at a pH of 7.0 or more and a pH of less than 8.0.

The mutant may be constructed by treating an existing mutagen or using the CRISPR gene scissors technology without introducing an exogenous DNA into a wild-type strain which is not a gene recombinant mutant through introduction of an exogenous gene to directly introduce RGEN RNPs into a target sequence in a ZEP gene.

The Chlamydomonas reinhardtii mutant of the present invention can accumulate a pigment, particularly, a xanthophyll-based pigment in the cells, and can include zeaxanthin in an even higher content among the pigments, so that the alga is cultured, and thus can be effectively used as a raw material for a food, feed, a medicine, and the like.

In this regard, the present invention relates to a culture of the Chlamydomonas reinhardtii mutant.

In the present invention, “a culture” refers to a medium in which a specific microorganism is cultured, that is, a post-culture medium, and the culture refers to a culture including the Chlamydomonas reinhardtii mutant. Further, the culture refers to a culture including all of the concentrate of the culture where a post-culture medium is subjected to processing such as concentration and drying, or the dry material of the culture. The culture can include a byproduct thereof, the preparation thereof is not limited, and as an example, the culture may be a liquid or a solid.

In order to culture a specific microorganism, the medium contains nutritional materials required by a subject to be cultured, that is, a microorganism to be cultured, and may be a medium in which a material for a special purpose is additionally added and mixed. The medium also refers to a culture medium or a culture solution, and is a concept encompassing all of the natural medium, the synthetic medium or the selective medium. A pH of the medium may be more than a range in which a Chlamydomonas reinhardtii mutant can be grown, and may be a pH of 6 or more as an example, and preferably a pH of 6 to 9.

Further, the present invention relates to a composition including one or more selected from the group consisting of the Chlamydomonas reinhardtii mutant of the present invention, a culture of the alga, a dry material thereof, and an extract thereof.

The composition may be used for improving the health of a human and an animal.

Since the mutant of the present invention has characteristics of producing a xanthophyll-based pigment including zeaxanthin and lutein and accumulating the pigment in vivo, the composition may be a pigment composition or a xanthophyll pigment composition in this regard.

The pigment composition may be a composition in which zeaxanthin is included in an amount of 5 to 15 parts by weight based on 100 parts by weight of the total pigments included in the composition. According to an exemplary embodiment of the present invention, as a result of measuring the content of zeaxanthin in the total pigments per each cell of the Chlamydomonas reinhardtii wild-type alga and the Chlamydomonas reinhardtii mutant, it could be confirmed that the Chlamydomonas reinhardtii mutant had a significantly high content of zeaxanthin in the pigment even when compared to the wild-type (FIG. 13C).

The pigment composition may be used as a raw material for a food or feed, and may be used as a preparation for oral administration.

Accordingly, a pigment composition or xanthophyll pigment composition including the composition or extract may be a composition for oral administration, in that the pigment composition or xanthophyll composition included in a food, a medicine, feed, or the like may be supplied via an oral route.

The composition for oral administration may be included in a formulated oral preparation by using a method publicly known in the art, such as a powder, a granule, a tablet, a pill, a sugar-coated tablet, a liquid, a gel, a syrup, a slurry, and a suspension. For example, for the oral preparation, a tablet or a purified material of sugar may be obtained by blending an active ingredient with a solid excipient, pulverizing the same, adding a suitable auxiliary agent thereto, and then processing the same into a granular mixture. Examples of a suitable excipient include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, and the like, starches including corn starch, wheat starch, rice starch, potato starch, and the like, celluloses including cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl cellulose, and the like, and fillers such as gelatin and polyvinylpyrrolidone. In addition, a crosslinked polyvinylpyrrolidone, agar, alginic acid, sodium alginate, or the like may be added as a disintegrating agent in some cases.

Furthermore, the composition can be added in order to achieve a purpose use which is special for a food or feed, and thus may be a food composition, a composition for a food additive, a feed composition or a composition for a feed additive. When the composition is used for feed or a food, health in the body can be maintained or strengthened by a xanthophyll pigment, particularly, zeaxanthin and lutein produced by the Chlamydomonas reinhardtii mutant and accumulated in the cells. Specifically, the zeaxanthin and lutein can prevent or alleviate degeneration of the macula, and the like as a macular pigment, and thus are effective for preventing or alleviating eye disorders related to the macular degeneration. More specifically, since the zeaxanthin and lutein have effects of strengthening or maintaining eye health; preventing or alleviating the macular degeneration; preventing or alleviating deterioration in eye function; alleviating or preventing damage to the retina; suppressing aging; maintaining retinal health; reducing the risk of developing the macular degeneration; or preventing or alleviating failing eyesight, the feed or food composition may be used for the use of preventing or alleviating the symptoms or the effects.

In the present invention, “for an additive” includes all food compositions as long as the food composition is a constitution in which ingredients other than the main ingredient are added to a food or feed, and a specific example thereof may be an effectively active material having functionality in a food or feed or a food additive defined by the Ministry of Food and Drug Safety of Republic of Korea to be added for coloring, preservation, and the like in a processed food.

The food may be a health functional food. More specifically, the food may be a food functional food for eye health.

The food, the food additive, the feed or the composition for a feed additive may further include other effective ingredients within a range not impairing the activity of the Chlamydomonas reinhardtii mutant of the present invention, a culture of the mutant, a dry material thereof, and an extract thereof. Further, it is possible to further include an additional ingredient such as a carrier.

In the present invention, a composition for feed may be prepared in the form of fermented feed, blended feed, a pellet, silage, and the like. The fermented feed may be prepared by including the Chlamydomonas reinhardtii mutant of the present invention, a dry fungus body of the mutant, a culture of the mutant, and an extract thereof, and additionally include various microbial bacteria or enzymes. The blended feed may be prepared by including various types of general feed, the mutant of the present invention, a dry fungus body of the mutant, a culture of the alga, and an extract thereof and mixing the mixture. A feed in the form of a pellet may be prepared by formulating the fermented feed or blended feed with a pellet machine. The silage may be prepared by mixing silage with a Chlamydomonas reinhardtii mutant, a dry fungus body of the mutant, a culture of the mutant and/or an extract thereof, but the use of the composition of the present invention is not limited thereto.

The composition may be mixed with a carrier and a flavoring typically used in the food or pharmaceutical field and may be prepared and administered in the form of a tablet, a troche, a capsule, an elixir, a syrup, a powder, a suspension, a granule, or the like. As the carrier, it is possible to use a binder, a lubricant, a disintegrating agent, an excipient, a solubilizing agent, a dispersing agent, a stabilizing agent, a suspending agent, and the like. As an administration method, an oral, parenteral, or application method may be used, but preferably, it is preferred that the composition is orally administered. In addition, an administration dose may be appropriately selected depending on the absorption degree, the inactivation rate and the excretion rate of an active ingredient in the body, and age, gender, status, and the like of a person to be treated. A pH of the composition can be easily changed depending on the production conditions and the like of medicine, food, and the like in which the composition is used.

The composition may include any one selected from the group consisting of a Chlamydomonas reinhardtii mutant, a culture of the mutant, a dry material thereof, and an extract thereof in an amount of 0.001 to 99.99 wt %, preferably 0.1 to 99 wt %, based on the total weight of the composition, and the content of an active ingredient may be appropriately adjusted depending on the method for using the composition and the purpose of using the composition.

The Chlamydomonas reinhardtii mutant may be included as it is or in a dried form in the composition, and the culture of the alga may be included in a concentrated or dried form in the composition. Furthermore, the dry material refers to a dried form of the alga or the culture thereof, and may be in the form of a powder prepared by lyophilization, and the like.

Further, the extract refers to an extract obtained by extracting a product from the Chlamydomonas reinhardtii mutant of the present invention, a culture solution thereof, or a dry material thereof, and includes an extract using a solvent, and the like, and an extract obtained by crushing the Chlamydomonas reinhardtii mutant of the present invention. Specifically, the extract may be an extract obtained by extracting and separating a pigment accumulated in the cells of the Chlamydomonas reinhardtii mutant of the present invention by a physical or chemical method.

The extraction procedure may be carried out by a typical method, and as an example, a target pigment may be extracted by adding an extraction solvent to the Chlamydomonas reinhardtii mutant of the present invention, homogenizing the resulting mixture, and then crushing the fungus body. After the extraction, a crushed material of the alga may be removed through centrifugation, and the extraction solvent may be removed by a method such as distillation under reduced pressure. In addition, the extraction procedure may further include a typical purification process. The aforementioned pigment has a property of being insoluble in water, and thus can be more easily extracted from the alga of the present invention.

Since the Chlamydomonas reinhardtii mutant of the present invention has an excellent ability to produce a xanthophyll, particularly zeaxanthin at low light intensity, a compound including the mutant and a byproduct thereof has effects of improving body activity, maintaining body functionality, and preventing deterioration in body functionality. Specifically, since the xanthophyll pigment is known to have an effect of suppressing macular degeneration, antioxidant and anticancer effects, and the like, the composition of the present invention may be used as a raw material included in a food, a health functional food, a medicine, feed, and the like for the use of maintaining body health, specifically, maintaining the body function with which the xanthophyll pigment is associated, preventing deterioration in body function, or improving the body function.

Furthermore, another object of the present invention is to provide a method for preparing a pigment using the Chlamydomonas reinhardtii mutant of the present invention.

Further, still another object of the present invention is to provide a method for preparing a food or feed raw material, including: culturing the Chlamydomonas reinhardtii mutant of the present invention.

When the Chlamydomonas reinhardtii mutant of the present invention is used, an amount of xanthophylls accumulated in the algae to be cultured may be increased, so that the supply of a raw material industrially used, and the like may be efficiently carried out.

The preparation method may include culturing the Chlamydomonas reinhardtii mutant of the present invention.

In addition, the preparation method may further include: separating the Chlamydomonas reinhardtii mutant of the present invention from the culture, after the culturing of the Chlamydomonas reinhardtii mutant of the present invention. The separated algae may be further subjected to a processing step including drying.

Furthermore, the preparation method may further include: extracting a pigment from the Chlamydomonas reinhardtii mutant of the present invention, a concentrate of the culture, or a dry material of the culture.

The culturing may be carried out in a medium under a pH condition of 6.0 to 8.0. Further, the culturing may be carried out under weak lighting conditions, specifically, under light intensity conditions within a range of 10 to 2,000 μmol photons/m²s. The Chlamydomonas reinhardtii mutant of the present invention has an excellent ability to produce a pigment even at low light intensity, and thus may increase the content of xanthophylls in the body, so that an excellent accumulation of xanthophylls may be achieved without inputting energy at high light intensity, and as a result, the Chlamydomonas reinhardtii mutant of the present invention may be industrially and effectively used.

The extraction may be carried out by a typical method such as a method for extracting a pigment from microorganisms, and examples thereof include an enzyme method, an ultrasonic extraction method, a mechanical extraction method, and the like, and are not limited thereto.

The preparation method may further include, in addition to the culturing step, a concentrating step of increasing the content of the alga after the culturing and a drying step of drying the alga subjected to the concentrating step by further reducing moisture in the alga. However, the concentrating step or the drying step is not necessarily needed, and may be generally carried out by using a concentrating and drying method, and a machine typically used in the field to which the present invention belongs.

The preparation method may be carried out by further including a purification step after the extracting step, and the purification step may be carried out by a typical purification method in the field to which the present invention belongs.

Xanthophylls prepared through the concentrating or drying step may be used as a raw material for a food, a health functional food, a cosmetic, a medicine, or the like.

The method for preparing xanthophylls may be carried out by adopting other methods within a range not impairing the effects of the present invention.

The contents on the mutant and the composition may also be applied mutatis mutandis to the preparation method of the present invention.

Hereinafter, the present invention will be described in detail through the Examples. However, the following Examples are only for exemplifying the present invention, and the scope of the present invention is not limited to the following Examples. The present Examples are provided to make the disclosure of the present invention perfect and to make a person skilled in the art to which the present invention belongs perfectly comprehend the scope of the present invention, and the present invention is defined only by the scope of the claims.

EXAMPLES Example 1: Construction of ZEP Gene Knock-Out Mutant

In order to target a ZEP gene of Chlamydomonas reinhardtii (phytozome: Cre02.g082550 or NCBI: AY211267.1) [phytozome.igi.doe.gov/pz/portal.html#!gene?search=1&detail=1&method=4614&searchText=transcriptid:30785220, present at a position of 1244277-1250969 in chromosome #2], for an sgRNA, five sgRNAs which allow the induction of a microhomology-driven frameshift mutation by using Cas-Designer (www.rgenome.net) were designed, and were synthesized through an in vitro transcription method. FIG. 3 is a description on the target sequences of the five sgRNAs constructed for targeting the ZEP gene. A Cas9 protein was prepared by expressing a recombinant Cas9 protein using E. coli, and performing purification. A Chlamydomonas reinhardtii cw15 mt-wild-type (CC-4349) used in the experiment was secured through the Chlamydomonas Resource Center (chlamycollection.org) [www.chlamycollection.org/product/cc-4349-cw15-mt-goodenough-330a/]. The Chlamydomonas cells were put into a 25° C. 50-ml flask containing a TAP medium [see Table 2], and were cultured while being irradiated with light using a fluorescence lamp at a light intensity of 70 uE and being shaken at 90 rpm. The concentration of the cells was measured by using a spectrophotometer, and cells during a period of actively culturing to an OD₇₅₀ of approximately 0.3 to 0.5 were used. In order to make a complex of RNPs, 200 ug of the Cas9 protein (FIG. 5, SEQ ID NO: 9) was mixed with 140 ug of the sgRNA (SEQ ID NO: 8) in nuclease-free water, and incubated at room temperature for 10 minutes. After the complex of the bound RNPs was transformed along with 50×10⁴ Chlamydomonas reinhardtii cw15 mt-wild-type (CC-4349) cells through (voltage 600 V, Capacity 50 μF) electric shock in a 4 mm electroporation cuvette by using a Biorad Gene Pulser Xcell™ electroporation system, the complex was subjected to gDNA extraction after dark incubation for 12 hours, and analyzed by performing targeted deep sequencing, and a single colony was obtained by diluting a part of the complex with 2,000 cells and streaking and spreading the diluted solution on a TAP agar plate. FIG. 4 is a result identifying the mutation of the ZEP gene, which was induced by RGEN-RNPs due to the targeted deep sequencing. The mutation of a target gene was identified by separating the single colony induced by the third sgRNA (0.456%) where the transformation efficiency was at the highest level. (Panel b of FIG. 4 illustrates the data of the targeted deep sequencing and of an experiment in which when the entire cells are collected and gDNAs are extracted and analyzed after the transformation experiment using the RNPs, all the mutations occurring in the DNA strands of a target site for the entire cells are analyzed, and the pattern and frequency thereof can be seen. That is, panel b of FIG. 4 illustrates patterns of the mutation actually identified at the target site through the targeted deep sequencing, but it is difficult to find out a big size change such as insertion of 42 bp or more by a principle of the targeted deep sequencing, and there may be a difference from a single colony actually obtained)

After the ZEP specific knock-out mutant was produced in this manner by using DNA-free RGEN RNPs, the Chl fluorescence with respect to all the cells was measured in the petri dish, and several estimated ZEP knock-out cell lines were selected. A circular shape indicates an estimated ZEP knock-out mutant grown on a TAP agar medium under dim light (50 μmol photons/m²s) conditions [Panel a of FIG. 4]. NPQ/4 images were measured by Imaging PAM (Walz). Unicellular colonies of the wild-type (WT) and ΔZEP mutant cell lines were grown on a minimum agar medium under dim light (50 μmol photons/m²s) conditions [Panes a and b of FIG. 6]. Among the thus-identified colonies, three mutants (ΔZ1, ΔZ2, and ΔZ3) where the content of the macular pigment was increased were selected, and a change in target DNA sequence at the actual position of the ZEP position was identified from the three ZEP mutants generated by RGEN RNPs through Sanger sequencing [FIG. 7].

As illustrated in FIG. 6b , it could be confirmed that the colonies of the Chlamydomonas reinhardtii cw15 wild-type and the mutants ΔZ1, ΔZ2, and ΔZ3 were grown in forms and sizes similar to one another on the TAP agar plate under the same light intensity conditions. Further, in the case of the cells liquid-cultured through photosynthesis in an HS medium, it can be confirmed that at the same concentration of the cells, the Chlamydomonas reinhardtii wild-type exhibits a striking green color, whereas the mutants ΔZ1, ΔZ2, and ΔZ3 exhibit a green color similar to a grass color tone [panel c of FIG. 6].

Among the selected mutants, the mutant Z1 was named as Chlamydomonas reinhardtii ZEP mutant 1 (ΔZ1), and the mutant was deposited at the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience & Biotechnology on Mar. 22, 2017 and given Accession No. KCTC 13230BP.

Example 2: Culturing of Mutant

1) Autotrophic Culture

In the case of an autotrophic culture in which the mutant was cultured in a state where an external carbon source was not supplied and by using only photosynthesis, the mutant was cultured in an HS medium which is a minimum medium by supplying 5% CO₂. After a medium having the composition as in the following Table 1 was produced, the medium was autoclaved and prepared, and the growth was initiated by making the concentration become 10⁶ cells/mL in the culture solution using cells in an active growth stage. A culture vessel was supplied with bubbles from beneath using a column made of glass as in FIG. 9, and was irradiated with light using a fluorescence lamp at a light intensity of 200 uE from both sides.

TABLE 1 HS Media Ingredients Con. in culture solution (mM or μM)) Buffer and Major Ingredients (mM) NH₄Cl 9.345 MgSO₄ • 7H₂O 0.08 CaCl₂ • 2H₂O 0.07 K₂HPO₄ 8.265 KH₂PO₄ 5.29 Trace Ingredients (μM) ZnSO₄ • 7H₂O 765 H₂BO₃ 922 MnCl₂ • 4H₂O 511 CoCl₂ • 6H₂O 7 CuSO₄ • 5H₂O 126 (NH₄)₆Mo₇O₂₄ • 4H₂O 18 FeSO₄ • 7H₂O 18 EDTA disodium salt 134 Others Carbon Source 5% CO₂ bubble, 80 cc/min pH in Culture Solution 7.0 Light Intensity 200 uE

2) Mixotrophic Culture

In the case of performing a mixotrophic culture where the mutant was cultured by supplying both photosynthesis and a carbon source, the mutant was cultured by adding acetic acid to a TAP medium. After a medium having the composition as in the following Table 2 was produced, the medium was autoclaved and prepared, and the growth was initiated by making the concentration become 10⁶ cells/mL in the culture solution using cells in an active growth stage. For the culture vessels, the mutant was cultured at large volumes by using a flask or bottle made of glass as in FIG. 10, and was stirred by using a magnetic bar. The mutant was together irradiated with light using a fluorescence lamp at a light intensity of 70 uE.

TABLE 2 TAP Media Ingredients Con. in culture solution (mM or μM)) Buffer and Major Ingredients (mM) NH₄Cl 7.5 CaCl₂ • 2H₂O 0.675 MgSO₄ • 7H₂O 0.8 K₂HPO₄ 0.62 KH₂PO₄ 0.41 Trace Ingredients (μM) EDTA • 2H₂O 135 FeSO₄ • 7H₂O 18 ZnSO₄ • 7H₂O 75 H₃BO₃ 185 MnCl₂ • 4H₂O 26 CuCl₂ • 2H₂O 6.5 Na₂MoO₄ • 2H₂O 5.5 CoCl₂ • 6H₂O 8.5 Others Carbon Source Glacial acetic acid, 1 ml/L Tris 2.42 g/L pH in Culture Solution 7.8 Light Intensity 70 uE

Example 4: Pigment Analysis of Mutant and Identification of Growth Characteristics

1) Pigment Analysis of Mutant

After a separation into a single colony as in Example 1, the mutant was continuously cultured, and a pigment analysis of each colony was carried out by using HPLC.

Specifically, the separated single colonies were cultured in a TAP medium under 70 μmol photons/m²s conditions for 3 days, and the specific culture conditions were carried out as under the culture conditions in 2) of Example 2. From the harvested alga, a pigment was extracted by using 80% acetone, and a centrifuged supernatant was filtered again by using a nylon filter, and then injected into an HPLC and analyzed.

Specifically, in order to separate the pigment, the total flow rate of the solvent was set at 1.2 mL per minute, and Tris with a pH of 8.0 and acetonitrile were each uniformly decreased from 14% and 84% to 0% from the 0th minute to the 15th minute, and methanol and ethyl acetate were increased starting from 2% to 68% and 32%, respectively, up to the 15th minute. Thereafter, this solvent ratio was maintained as it was for 3 minutes (from the 15th minute to the 18th minute), and then the ratio of each solvent was returned to the ratio at the start for 1 minute (from the 18th minute to the 19th minute), and then a post-run was performed while maintaining the solvent ratio as it was for the remaining 6 minutes. Shimadzu LC-20A Prominence manufactured by Shimadzu Company was used as a pump, Watera Spherisorb TMS5 (DS1 4.6×250 mm, 5 μm Cartridge Column, USA) was used as a column, and the temperature of the column was maintained at 40° C. Data was analyzed by using a photodiode array detector (SPD-M20A, Shimadzu) as a detector, and the concentrations were obtained by using a standard curve which quantified a carotenoid and chlorophylls a and b purchased from Agern Alle, Horsholm, Denmark (DHL) as a standard from the result in which carotenoid pigments including zeaxanthin and chlorophyll a were detected at 445 nm and 670 nm, respectively.

FIG. 11 illustrates HPLC analysis graphs illustrating a pigment profile of each alga, and FIG. 13 illustrates a set of graphs which quantitatively analyze the contents of zeaxanthin and lutein of each alga grown under 200 μmol photons/m²s using HPLC.

2) Identification of Growth Rate

In order to compare the cell proliferation rates and final growth amounts of the wild-type Chlamydomonas reinhardtii alga and the mutants ΔZ1, ΔZ2, and ΔZ3, the alga and the mutants were cultured under light intensity conditions of 200 μmol photons/m²s in a state where 5% CO2 bubbles were supplied in an HS medium which is a minimum photosynthesis medium [see Table 1]. The number of initially inoculated cells was 1×10⁶ cells/ml, and a growth curve was drawn by measuring the number of cells at intervals of 12 hours for 60 hours. FIG. 12 is an experimental result of comparing the cell growth rates through photosynthesis using carbon dioxide in the wild-type (WT) and the ZEP knock-out mutants. As illustrated in FIG. 12, it was confirmed that the mutants ΔZ1, ΔZ2, and ΔZ3 had a growth rate at a level similar to the wild-type as the culture period elapsed.

Example 5: Identification of Pigment Production

The contents of lutein and zeaxanthin pigments of the cells were quantitatively analyzed at intervals of 12 hours by using HPLC simultaneously while carrying out the experiment in FIG. 12. Through FIG. 13, the amounts of lutein and zeaxanthin produced from the Chlamydomonas reinhardtii cw15 wild-type (WT) and the ZEP knock-out mutants over time were compared with one another. When the results of the ZEP knock-out mutants were compared to those of the wild-type, it could be confirmed that the amounts of lutein produced from the ZEP knock-out mutants were at a level similar to that of the wild-type, but the amounts of zeaxanthin insignificantly present in the wild-type were greatly increased by at least 50 times or more.

The following Table 3 and FIG. 14 compare the contents of zeaxanthin and lutein from higher plants known to have high contents of zeaxanthin and lutein with those from the Chlamydomonas reinhardtii cw15 wild-type (WT) and the ZEP knock-out mutants. For the Chlamydomonas, the content (μg/100 g) was calculated by dividing the amounts of lutein and zeaxanthin pigments by the dry weight of the cells at 36 hours in FIG. 12. The pigment contents (μg/100 g) of the remaining higher plants known to have high contents of zeaxanthin and lutein were compared by citing the USDA National Nutrient Database for Standard Reference, Release 23 (2010). When the contents of lutein and zeaxanthin were compared with those of the higher plants, the Chlamydomonas reinhardtii wild-type exhibited a content which is higher by at least 6 times or more (than that of nasturtium) and the ZEP gene knock-out mutants exhibited a content which is higher by 12 times or more (than that of nasturtium). In particular, when the content of zeaxanthin was compared with those of the higher plants, the ZEP gene knock-out mutants exhibited a content which is higher by at least 120 times or more (than that of orange pepper).

Through FIGS. 13 and 14, high productivities and contents were identified, and through this, it could be confirmed that the mutants had high competitiveness in the raw material market of the lutein and zeaxanthin pigment industry.

TABLE 3 Lutein + Zeaxanthin Product (μg/100 g) Chlamydomonas, cw15-, WT 274,397 Chlamydomonas, ZEP mutant 1 528,353 (ΔZ1) Chlamydomonas, ZEP mutant 2 502,520 (ΔZ2) Chlamydomonas, ZEP mutant 3 544,684 (ΔZ3) Nasturtium (yellow flowers) 45,000 Kale (raw) 39,550 Kale (cooked) 18,246 Dandelion leaves (raw) 13,610 Nasturtium (leaves) 13,600 Turnip greens (raw) 12,825 Spinach (raw) 12,198 Spinach (cooked) 11,308 Swiss chard (raw or cooked) 11,000 Turnip greens (cooked) 8440 Collard greens (cooked) 7694 Watercress (raw) 5767 Garden peas (raw) 2593 Romaine lettuce 2312 Zucchini 2125 Brussels sprouts 1590 Pistachio nuts 1205 Broccoli 1121 Carrot (cooked) 687 Maize/corn 642 Egg (hard boiled) 353 Avocado (raw) 271 Carrot (raw) 256 Kiwi fruit 122

It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided they come within the scope of the appended claims and their equivalents.

[Information on Deposit of Microorganism]

Name of Depository Institution: Korea Research Institute of Bioscience & Biotechnology

Accession number: KCTC13230BP

Deposit date: Mar. 22, 2017

Chlamydomonas reinhardtii ZEP mutant 1 (ΔZ1) (accession number: KCTC 13230BP) was deposited with Korea Research Institute of Bioscience and Biotechnology, on Mar. 22, 2017. The subject strain has been deposited under conditions that assure that access to the strain will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be 10 available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject deposits will be stored and made available to the public in accord with 15 the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit 20 should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it. 

The invention claimed is:
 1. A Chlamydomonas reinhardtii mutant having a ZEP gene mutation in which a base sequence represented by SEQ ID No. 4 is inserted between a 816th base and a 817th base in a ZEP gene sequence of a Chlamydomonas reinhardtii represented by SEQ ID No. 1 and having an ability to produce a xanthophyll.
 2. The Chlamydomonas reinhardtii mutant of claim 1, wherein the Chlamydomonas reinhardtii mutant has a ZEP gene mutation represented by SEQ ID No.
 5. 3. A composition comprising one or more selected from the group consisting of the Chlamydomonas reinhardtii mutant of claim 1, a culture thereof, a dried material thereof, and an extract thereof. 